Methods and compositions for modulating the interaction between the APJ receptor and the HIV virus

ABSTRACT

The orphan seven transmembrane domain receptor, APJ, can function as a coreceptor for cellular infection by the HIV virus. The establishment of cell lines that coexpress CD4 and APJ provide valuable tools for continuing research on HIV infection and the development of anti-HIV therapeutics.

BACKGROUND OF THE INVENTION

[0001] The entry of HIV-1 into cells involves binding of the viralenvelope (env) protein to CD4 followed by interaction with one ofseveral coreceptors (reviewed in E. A. Berger, 1997, AIDS, 11:S3-S16;Broder et al., 1997, J. Leukocyte Biol., 62:20-29; Doms et al., 1997,Virology, 235:279-190; and Moore et al., 1997, Curr. Opinion Immunol.,9:551-562). Binding of the env protein to the appropriate coreceptor isthought to trigger conformational changes in env that mediate fusionbetween the viral membrane and the host cell membrane. The CCR5 andCXCR4 chemokine receptors have been identified as major HIV-1coreceptors in that all HIV-1 strains examined to date use one or bothof these molecules as second receptors. CCR5 supports infection by R5(M-tropic) virus strains, while CXCR4 supports infection by X4(T-tropic) virus isolates (Alkhatib et al., 1996, Science,272:1955-1958; Berger et al., 1998, Nature, 391:240; Choe et al., 1996,Cell, 85:1135-1148; Deng et al., 1996, Nature, 381:661-666; Doranz etal., 1996, Cell, 85:1149-1158; Dragic et al., 1996, Nature, 381:667-673;and Feng et al., 1996, Science, 272:872-877). R5-X4 (dual-tropic) viralenv proteins can, in conjunction with CD4, use either CCR5 or CXCR4 forcellular entry. The differential utilization of CCR5 and CXCR4 by HIVstrains, coupled with their expression patterns in CD4 positive cellslargely explains viral tropism at the level of entry.

[0002] In addition to CCR5 and CXCR4, a number of other chemokine andorphan seven transmembrane domain receptors have been shown to functionas coreceptors for one or more virus strains in vitro, including CCR2b,CCR3, CCR8, CX3CR1, GPR1, GPR 15, STRL33, US28, and ChemR23 (Choe etal., 1996, Cell 85:1135-1148; Deng et al., 1997, Nature 388:296-300;Doranz et al., 1996, Cell 85:1149-1158; Farzan et al., 1997, J. Exp.Med. 186:405-411; Liao et al., 1997, J. Exp. Med. 195:2015-2023;Pleskoffet al., 1997, Science 276:1874-1878; Reeves et al., 1997,Virology 231:120-134; Rucker et al., 1997, J. Virol. 71:8999-9007). Ingeneral, these alternative coreceptors support virus infection lessefficiently than either CCR5 or CXCR4. However, use of alternativecoreceptors may help explain certain facets of HIV-1 tropism andpathogenesis in vivo. For example, neurologic disease is a serious andrelatively frequent consequence of HIV-1 infection, with microglia beingthe primary targets of virus infection in the central nervous system(Bagasra et al., 1996, AIDS, 10:573-585; Sharer et al., 1992, J.Neuropath. Exp. Neurol., 51:3-11; Wiley et al., 1986, Proc. Natl. Acad.Sci., USA, 83:7089-7093). Microglia express both CCR3 and CCR5 and ithas been suggested that utilization of CCR3 by a virus strain maycorrelate with neurotropism (He et al., 1997, Nature, 385:645-649).

[0003] The identification of additional coreceptors for the HIV viruswould provide an important tool for investigating and controlling HIVinfection.

SUMMARY OF THE INVENTION

[0004] In one aspect of the invention, the invention relates to arecombinant eukaryotic cell transformed with a polynucleotide encodingan APJ polypeptide and/or a polynucleotide encoding a CD4 polypeptide,wherein the cell coexpresses APJ and CD4 polypeptides.

[0005] The invention also relates to an antibody which specificallybinds to an extracellular domain of APJ, wherein the antibody inhibitsHIV infection of a target cell that coexpresses APJ and CD4 polypeptidesor wherein the antibody inhibits membrane fusion between a first cellcoexpressing APJ and CD4 polypeptides and a second cell expressing anHIV env protein.

[0006] The invention also relates to a substantially purified peptidefragment of APJ, wherein the peptide inhibits HIV infection of a targetcell that coexpresses APJ and CD4 polypeptides or wherein the peptideinhibits cell fusion between a first cell coexpressing APJ and CD4polypeptides and a second cell expressing an HIV env protein.

[0007] In another aspect of the invention the invention relates tomethods for identifying compounds that modulate the interaction betweenan HIV virus and an APJ receptor.

[0008] The invention also relates to a method of inhibiting HIVinfection of a target cell expressing an APJ and CD4 polypeptidescomprising contacting the target cell with an effective amount of an APJbinding or blocking agent.

[0009] The invention also relates to a method of treating a subjecthaving or at risk of having an HIV infection or related disorder,comprising administering a therapeutically effective amount of ananti-APJ antibody to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a graph showing cell-cell fusion mediated by HIV-1 orHIV-2 env proteins. All of the indicated env proteins are derived fromHIV-1, with the exception of HIV-2/ST which was derived from HIV-2. QT6cells expressing CD4, the indicated coreceptor, and luciferase under thecontrol of the T7 promoter were mixed with cells expressing T7polymerase and the indicated HIV-1 or HIV-2 env protein. The degree ofcell-cell fusion was determined 8 hours post-mixing by measuringluciferase activity. The results were normalized by setting the extentof fusion obtained when CD4 and either CCR5 (for R5 env proteins) orCXCR4 (for X4 env proteins) were coexpressed to 100%. The extent offusion obtained with the major HIV-1 coreceptors was generally 40 to 100times above background levels. Error bars here and in subsequent figuresrepresent the standard error of the mean derived from multipleindependent experiments.

[0011]FIG. 2 is a graph showing cell-cell fusion mediated by SIV envproteins. Cell-cell fusion was determined as in FIG. 1, but using SIVrather than HIV env proteins. The results were normalized by setting theextent of fusion obtained when CD4 and CCR5 were coexpressed to 100%.The extent of fusion obtained with the different env proteins wasgenerally 40 to 100 times above background levels (defined as CD4alone).

[0012]FIG. 3 is a graph showing pseudotype virus infection. HEK 293cells expressing CD4 and the indicated coreceptor were infected withluciferase virus pseudotypes bearing the indicated HIV or SIV envprotein, and luciferase activity was determined 2-3 days afterinfection.

[0013]FIG. 4 is a graph showing viral infection of HEK 293 cells thatexpress CD4 and the indicated coreceptor and that also contain a plasmidencoding luciferase under the control of the HIV-1 LTR. The cells wereinfected with live HIV-1 IIIB (HxB) or HIV-1 89.6. Values werenormalized by setting the extent of infection obtained with either CCR5(R5 env proteins) or CXCR4 (X4 env proteins) to 100%.

[0014]FIG. 5 is an image of a Southern blot showing the entry of virusinto cells expressing CD4 and APJ. QT6 cells stably expressing CD4 andtransiently expressing the desired coreceptor were infected withDNAase-treated, cell-free virus. Viral specific LTR DNA sequences weredetected 2 days after infection by PCR amplification, followed byresolution of the products on a 2% agarose gel and detection ofsequences using a labeled probe.

[0015]FIG. 6 is an image of a Northern blot showing the expression ofAPJ in human brain tissue. Membranes containing poly A⁺ RNA from varioushuman brain regions were obtained from Clontech and were incubated witha labeled cDNA probe specific for APJ overnight. The membranes were thenexposed to a Fuji Imaging plate for 4 hours. The following tissues wereexamined: Panel (A): (1) Amygdala; (2) Caudate nucleus; (3) Corpuscallosum; (4) Hippocampus; (5) Whole brain; (6) Substantia nigra; (7)Subthalamic nucleus; (8) Thalamus; Panel (B): (1) Cerebellum; (2)Cerebral cortex; (3) Medulla; (4) Spinal cord; (5) Occipital lobe; (6)Frontal lobe; (7) Temporal lobe; (8) Putamen.

[0016]FIG. 7 is an image of a Northern blot showing the expression ofAPJ in human peripheral tissue. Membranes containing poly A⁺ RNA fromvarious human tissues were obtained from Clontech and were incubatedwith a labeled cDNA probe specific for APJ overnight. The membranes werethen exposed to a Fuji Imaging plate for 4 hours. The following tissueswere examined: (1) Spleen; (2) Thymus; (3) Prostate; (4) Testis; (5)Ovary; (6) Small intestine; (7) Colonic mucosa; (8) Total peripheralblood lymphocytes.

[0017]FIG. 8 is an image of a stained agarose gel showing the expressionof APJ in primary cells and in T-cell lines. RNA from the indicatedcells was used in one-tube RT-PCR reactions and 10 μl of each 25 μlreaction was run out on a 2% agarose gel. The size of the predicted APJband is 481 base pairs. Both plasmid DNA and RNA isolated from U87-APJstably transfected cells are included as positive controls and water wasused as template for a negative control.

[0018]FIG. 9 is the nucleotide and deduced amino acid sequence for humanAPJ (O'Dowd et al., 1993, Gene 136:355-360) (SEQ ID NO: 1). Thepositions of the seven trans-membrane regions are as follows:transmembrane region 1 (TM 1) corresponds to amino acids 26-54;transmembrane region 2 (TM2) corresponds to amino acids 66-90;transmembrane region 3 (TM3) corresponds to amino acids 104-125;transmembrane region 4 (TM4) corresponds to amino acids 144-167;transmembrane region 5 (TM5) corresponds to amino acids 199-225;transmembrane region 6 (TM6) corresponds to amino acids 246-271;transmembrane region 7 (TM7) corresponds to amino acids 285-312.Extracellular portions of the APJ polypeptide are located in the aminoterminal segment before transmembrane domain 1 (e.g. amino acids 1-25),between transmembrane domains 2 and 3 (e.g. amino acids 91-103), betweentransmembrane domains 4 and 5 (e.g. amino acids 168-198), and betweentransmembrane domains 6 and 7 (e.g. amino acids 272-284).

DETAILED DESCRIPTION OF THE INVENTION

[0019] In accordance with this invention, it has been discovered thatthe orphan seven transmembrane domain receptor, APJ (O'Dowd et al.,1993, Gene 136:355-360), functions as an efficient coreceptor for anumber of HIV-1 and SIV strains. APJ serves as a very efficientcoreceptor for some X4 (T-tropic) and R5-X4 (dual tropic) virus strains,while two R5 (M-tropic) isolates use APJ less efficiently. APJ alsoserved as a coreceptor for several SIV strains.

[0020] Also in accordance with this invention, the widespread expressionof APJ in the human central nervous system has been discovered. Theefficient use of APJ by a number of virus strains, coupled with theexpression of APJ in the central nervous system, indicates thatutilization of this receptor may be important in HIV neuropathogenesis.

[0021] Also in accordance with this invention, the expression of APJ ina CD4 positive T-cell line, C8166, has been discovered.

[0022] In one embodiment, the present invention relates to recombinantcell lines, the cells of which co-express APJ and CD4 polypeptides, andwhich contain an exogenous polynucleotide that encodes either an APJpolypeptide or a CD4 polypeptide. The present invention also relates torecombinant cell lines, the cells of which co-express APJ and CD4polypeptides, and which contain an exogenous polynucleotide that encodesan APJ polypeptide and an exogenous polynucleotide that encodes a CD4polypeptide. As used herein, a “CD4 polypeptide” means a mammalian CD4polypeptide, preferably a human or a simian CD4 polypeptide, or abiologically active fragment thereof. As used herein, an “APJpolypeptide” means a mammalian APJ polypeptide, preferably a human or asimian APJ polypeptide, or a biologically active fragment thereof.Biologically active, as used herein, refers to polypeptides having anability to specifically interact with an HIV or SIV virus and topolypeptides having at least one epitope for an antibody immunoreactivewith an APJ or a CD4 polypeptide.

[0023] The invention relates not only to naturally-occurring APJ and CD4polypeptides, but also to mutant APJ and CD4 polypeptides. For example,changes in the amino acid sequence of APJ are contemplated in thepresent invention. APJ can be altered by changing the DNA encoding theprotein. Preferably, only conservative amino acid sequence alterationsare undertaken, using amino acids that have the same or similarproperties. Illustrative amino acid substitutions include the followingchanges: alanine to serine; arginine to lysine; asparagine to glutamineor histidine; aspartate to glutamate; cysteine to serine; glutamine toasparagine; glutamate to aspartate; glycine to proline; histidine toasparagine or glutamine; isoleucine to leucine or valine; leucine tovaline or isoleucine; lysine to arginine, glutamine, or glutamate;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucineor leucine.

[0024] Polynucleotide sequences of the invention include DNA, cDNA andRNA sequences which encode APJ or CD4 polypeptides. Such polynucleotidesinclude naturally occurring, synthetic, and intentionally manipulatedpolynucleotides. For example, portions of the mRNA sequence may bealtered due to alternate RNA splicing patterns or the use of alternatepromoters for RNA transcription. As another example, APJ and CD4polynucleotides may be subjected to site-directed mutagenesis. Thepolynucleotide sequence for APJ and CD4 also includes antisensesequences. The invention also includes a polynucleotide encoding an APJpolypeptide having biological activity or a CD4 polypeptide havingbiological activity.

[0025] Suitable cell types include, but are not limited to, cells of thefollowing types: NIH 3T3 (Murine), Mv 1 lu (mink), BS-C-1 (African GreenMonkey), human embryonic kidney (HEK) 293 cells (ATCC CRL 1573), andquail QT6 cells. Such cells are described, for example, in the Cell LineCatalog of the American Type Culture Collection (ATCC). These cells canbe stably transformed or transiently transformed by a method known tothe skilled artisan. See, for example, Ausubel, et al., Introduction ofDNA Into Mammalian Cells, in Current Protocols in Molecular Biology,sections 9.5.1-9.5.6 (John Wiley & Sons, Inc. 1995). “Stable”transformation in the context of the invention means that the cells areimmortal to the extent of having gone through at least 50 divisions.

[0026] Exogenous APJ or CD4 polynucleotides can be expressed usinginducible or constitutive regulatory elements for expression. Commonlyused constitutive or inducible promoters, for example, are known in theart. For example promoters derived from the genome of mammalian cells(e.g. metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat, the adenovirus late promoter, thevaccinia virus 7.5K promoter) may be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the exogenous APJ or CD4 polynucleotides.

[0027] The desired protein encoding sequence and operably linkedpromoter may be introduced into a recipient cell either as anon-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the desired molecule may occur through the transient expression ofthe introduced sequence. Alternatively, permanent expression may occurthrough the integration of the introduced sequence into the hostchromosome.

[0028] In a preferred embodiment, the introduced sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. A wide variety of vectors may beemployed for this purpose. Factors of importance in selecting aparticular plasmid or viral vector include the following: the ease withwhich recipient cells that contain the vector may be recognized andselected from those recipient cells which do not contain the vector; thenumber of copies of the vector which are desired in a particular host;and whether it is desirable to “shuttle” the vector between host cellsof different species.

[0029] Several possible vector systems are available for expression. Oneclass of vectors utilize DNA elements which provide autonomouslyreplicating extra-chromosomal plasmids, derived from animal viruses suchas bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus. Asecond class of vectors include vaccinia virus expression vectors. Athird class of vectors relies upon the integration of the desired genesequences into the host chromosome. Cells which have stably integratedthe introduced DNA into their chromosomes may be selected by alsointroducing one or more markers (e.g., an exogenous gene) which allowselection of host cells which contain the expression vector. The markermay provide, for example, prototrophy to an auxotrophic host or biocideresistance, e.g., antibiotic resistance or heavy metal resistance, suchas copper resistance. The selectable marker gene can either be directlylinked to the DNA sequences to be expressed, or introduced into the samecell by co-transformation. Additional elements may also be needed foroptimal synthesis of mRNA. These elements may include splice signals, aswell as transcription promoters, enhancers, and termination signals. ThecDNA expression vectors incorporating such elements include thosedescribed by Okayama, H., Mol. Cell. Biol., 3:280 (1983), and others.

[0030] Once the vector or DNA sequence containing the construct has beenprepared for expression, the DNA construct may be introduced(transformed) into an appropriate host. Various techniques which may beemployed include, for example, protoplast fusion, calcium phosphateprecipitation, electroporation, microinjection, delivery via liposomes,viral infection, or other conventional techniques.

[0031] In another embodiment, the present invention relates totransgenic animals having cells that coexpress human CD4 and APJpolypeptides. Such transgenic animals represent a model system for thestudy of HIV infection and the development of more effective anti-HIVtherapeutics.

[0032] The term “animal”, as used herein, denotes all mammalian speciesexcept human. It also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages. Farm animals (pigs,goats, sheep, cows, horses, rabbits, etc.), rodents (such as mice), anddomestic pets (for example, cats and dogs) are included within the scopeof the present invention.

[0033] A “transgenic” animal is any animal containing cells that beargenetic information received, directly or indirectly, by deliberategenetic manipulation at the subcellular level, such as by microinjectionor by infection with a recombinant virus.

[0034] “Transgenic” in the present context does not encompass classicalcrossbreeding or in vitro fertilization, but rather denotes animals inwhich one or more cells receive a recombinant DNA molecule. Although itis highly preferred that this molecule be integrated within the animal'schromosomes, the present invention also contemplates the use ofextrachromosomally replicating DNA sequences, such as might beengineered into yeast artificial chromosomes.

[0035] The term “transgenic animal” also includes a “germ cell line”transgenic animal. A germ cell line transgenic animal is a transgenicanimal in which the genetic information has been taken up andincorporated into a germ cell line, therefore conferring the ability totransfer the information to offspring. If such- offspring in factpossess some or all of that information they are also consideredtransgenic animals.

[0036] It is highly preferred that the transgenic animals of the presentinvention be produced by introducing into single cell embryos apolynucleotide encoding an APJ polypeptide and or a polynucleotideencoding a CD4 polypeptide, in a manner such that these polynucleotidesare stably integrated into the DNA of germ line cells of the matureanimal and inherited in normal mendelian fashion. Advances intechnologies for embryo micromanipulation now permit introduction ofexogenous polynucleotides into fertilized mammalian ova. For instance,totipotent or pluripotent stem cells can be transformed bymicroinjection, calcium phosphate mediated precipitation, liposomefusion, retroviral infection or other means, the transformed cells arethen introduced into the embryo, and the embryo then develops into atransgenic animal. In a preferred method, developing embryos areinfected with a retrovirus containing the desired polynucleotide, andtransgenic animals produced from the infected embryo.

[0037] In a most preferred method, the appropriate polynucleotides arecoinjected into the pronucleus or cytoplasm of embryos, preferably atthe single cell stage, and the embryos allowed to develop into maturetransgenic animals. These techniques are well known. For example,reviews of standard laboratory procedures for microinjection ofexogenous DNAs into mammalian (mouse, pig, rabbit, sheep, goat, cow)fertilized ova include: Hogan et al., Manipulating The Mouse Embryo(Cold Spring Harbor Press 1986); Krimpenfort et al., Bio/Technology 9:86(1991); Palmiter et al., Cell 41:343 (1985); Kraemer et al., GeneticManipulation of The Early Mammalian Embryo (Cold Spring HarborLaboratory Press 1985); Hammer et al., Nature 315:680 (1985); Purcel etal., Science 244: 1281 (1986); Wagner et al., U.S. Pat. No. 5,175,385;Krimpenfort et al., U.S. Pat. No. 5,175,384, the respective contents ofwhich are herein incorporated by reference.

[0038] The polynucleotide that encodes APJ or CD4 can be fused in properreading frame under the transcriptional and translational control of avector to produce a genetic construct that is then amplified, forexample, by preparation in a bacterial vector, according to conventionalmethods. See, for example, the standard work: Sambrook et al., MolecularCloning: a Laboratory Manual (Cold Spring Harbor Press 1989), thecontents of which are herein incorporated by reference. The amplifiedconstruct is thereafter excised from the vector and purified for use inproducing transgenic animals.

[0039] Production of transgenic animals containing the gene for humanCD4 have been described. See Snyder et al., Mol. Reprod. & Devel.40:419-428 (1995); Dunn et al., J Gen. Virology 76:1327-1336 (1995), thecontents of which are incorporated by reference.

[0040] In another embodiment, the present invention relates toantibodies that bind APJ and that inhibit HIV entry into a CD4-positivetarget cell or that inhibit cell-cell fusion between a first cell typethat expresses CD4 and APJ polypeptides and a second cell type thatexpresses the env protein. As used herein, an env protein means any envprotein derived from an HIV virus, either HIV-1 or HIV-2, or derivedfrom an SIV virus. Expression of an env protein by a cell will typicallyresult in the expression of the gp120 moiety of the env protein on thecell surface. Antibodies of the invention may also inhibit gp120 bindingto APJ. Such antibodies could represent research and diagnostic tools inthe study of HIV infection and the development of more effectiveanti-HIV therapeutics. In addition, pharmaceutical compositionscomprising antibodies against APJ may represent effective anti-HIVtherapeutics.

[0041] An antibody suitable for blocking env-mediated cell-cell fusion,HIV entry into a CD4 positive cell, or gp120 binding to APJ is specificfor at least one portion of an extracellular region of the APJpolypeptide, e.g. the first extracellular region (amino acids 1-25), thesecond extracellular region (amino acids 91-103), the thirdextracellular region (amino acids 168-198), or the fourth extracellularregion (amino acids 272-284), as shown in FIG. 9. Preferred antibodiesare those which recognize an epitope comprising a portion of either thefirst extracellular region or the second extracellular region.Particularly preferred antibodies are those which recognize an epitopecomprising the Asn-Tyr-Tyr-Gly (SEQ ID NO: 3) amino acid sequencecontained within the first extracellular region.

[0042] Anti-APJ antibodies of the invention include polyclonalantibodies, monoclonal antibodies, and fragments of polyclonal andmonoclonal antibodies. The preparation of polyclonal antibodies iswell-known to those skilled in the art. See, for example, Green et al.,Production of Polyclonal Antisera, in Immunochemical Protocols (Manson,ed.), pages 1-5 (Humana Press 1992); Coligan et al., Production ofPolyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CurrentProtocols in Immunology, section 2.4.1 (1992), which are herebyincorporated by reference.

[0043] The preparation of monoclonal antibodies likewise isconventional. See, for example, Kohler & Milstein, Nature 256:495(1975); Coligan et al., Current Protocols in Immunology, sections2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page726 (Cold Spring Harbor Pub. 1988), which are hereby incorporated byreference. Briefly, monoclonal antibodies can be obtained by injectingmice with a composition comprising an antigen, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B lymphocytes, fusing the B lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures. Monoclonal antibodies can be isolated andpurified from hybridoma cultures by a variety of well-establishedtechniques. Such isolation techniques include affinity chromatographywith Protein-A Sepharose, size-exclusion chromatograhy, antigen affinitypurification and ion-exchange chromatography. See, e.g., Coligan et al.,Current Protocols in Immunology, sections 2.7.1-2.7.12 and sections2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG), inMethods in Molecular Biology, Vol. 10, pages 79-104 (Humana Press 1992).

[0044] Methods of in vitro and in vivo multiplication of monoclonalantibodies are well-known to those skilled in the art. Multiplication invitro may be carried out in suitable culture media such as Dulbecco'sModified Eagle Medium or RPMI 1640 medium, optionally replenished by amammalian serum such as fetal calf serum or trace elements andgrowth-sustaining supplements such as normal mouse peritoneal exudatecells, spleen cells, bone marrow macrophages. Production in vitroprovides relatively pure antibody preparations and allows scale-up toyield large amounts of the desired antibodies. Large scale hybridomacultivation can be carried out by homogenous suspension culture in anairlift reactor, in a continuous stirrer reactor, or in immobilized orentrapped cell culture. Multiplication in vivo may be carried out byinjecting cell clones into mammals histocompatible with the parentcells, e.g., osyngeneic mice, to cause growth of antibody-producingtumors. Optionally, the animals are primed with a hydrocarbon,especially oils such as pristane (tetramethylpentadecane) prior toinjection. After one to three weeks, the desired monoclonal antibody isrecovered from the body fluid of the animal.

[0045] Therapeutic applications for anti-APJ antibodies disclosed hereinare also part of the present invention. For example, antibodies of thepresent invention may also be derived from subhuman primate antibody.General techniques for raising therapeutically useful antibodies inbaboons can be found, for example, in Goldenberg et al., InternationalPatent Publication WO 91/11465 (1991) and Losman et al., Int. J Cancer46:310 (1990), which are hereby incorporated by reference.

[0046] Alternatively, a therapeutically or diagnostically usefulanti-APJ antibody may be derived from a “humanized” monoclonal antibody.Humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and thensubstituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833 (1989),which is hereby incorporated in its entirety by reference. Techniquesfor producing humanized monoclonal antibodies are described, forexample, by Jones et al., Nature 321:522 (1986); Riechmann et al.,Nature 332:323 (1988); Verhoyen et al., Science 239:1534 (1988); Carteret al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Sandhu, Crit. Rev.Biotech. 12:437 (1992); and Singer et al., J. Immunol. 150:2844 (1933),which are hereby incorporated by reference.

[0047] Antibodies of the invention may also be derived from humanantibody fragments isolated from a combinatorial immunoglobulin library.See, for example, Barbas et al., Methods: A Companion to Methods inEnzymology, Vol. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol.12:433 (1994), which are hereby incorporated by reference. Cloning andexpression vectors that are useful for producing a human immunoglobulinphage library can be obtained, for example, from Stratagene CloningSystems (La Jolla, Calif.).

[0048] In addition, antibodies of the present invention may be derivedfrom a human monoclonal antibody. Such antibodies are obtained fromtransgenic mice that have been “engineered” to produce specific humanantibodies in response to an antigenic challenge. In this technique,elements of the human heavy and light chain loci are introduced intostrains of mice derived from embryonic cell lines that contain targeteddisruptions of the endogenous heavy and light chain loci. The transgenicmice can synthesize human antibodies specific for human antigens, andthe mice can be used to produce human antibody-secreting hybridomas.Methods for obtaining human antibodies from transgenic mice aredescribed by Green et al., Nature Genet. 7:13 (1994); Lonberg et al.,Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579 (1994),which are hereby incorporated by reference.

[0049] Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment.

[0050] Antibody fragments can be obtained by pepsin or papain digestionof whole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647, and references contained therein.These patents are hereby incorporated in their entireties by reference.See also Nisonhoff et al., Arch. Biochem. Biophys. 89:230 (1960);Porter, Biochem. J. 73:119 (1959); Edelman et al., Methods inEnzymology, Vol. 1, page 422 (Academic Press 1967); and Coligan et al.,Current Protocols in Immunology, sections 2.8.1-2.8.10 and2.10.1-2.10.4.

[0051] Other methods of cleaving antibodies, such as separation of heavyand light chains to form monovalent light-heavy chain fragments, furthercleavage of fragments, or other enzymatic, chemical, or genetictechniques may also be used, so long as the fragments bind to theantigen that is recognized by the intact antibody.

[0052] For example, Fv fragments comprise an association of V_(H) andV_(L) chains. This association may be noncovalent, as described in Inbaret al., Proc. Natl. Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu,Crit. Rev. Biotech. 12:437 (1992). Preferably, the Fv fragments comrpiseV_(H) and V_(L) chains connected by a peptide linker. These single-chainantigen binding proteins (sFv) are prepared by constructing a structuralgene comprising DNA sequences encoding the V_(H) and V_(L) domainsconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology, Vol. 2, page 97(1991); Bird et al., Science 242:423-426 (1988); Ladner et al., U.S.Pat. No. 4,946,778; Pack et al., Bio/Technology 11:1271-77- (1993); andSandhu, Crit. Rev. Biotech. 12:437 (1992).

[0053] Another form of an antibody fragment is a peptide coding for asingle complementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology, Vol. 2, page 106 (1991).

[0054] Antibodies that bind to the CXCR4 chemokine receptor, another HIVcoreceptor, have been shown to block fusion of HIV strains that useCXCR4 receptor for infection (Feng, et al., Science 272:872 (1996);Endres, et al., Cell 87:745 (1996)).

[0055] In another embodiment, the present invention relates to “APJvariants”. An APJ variant, as used herein, means a molecule thatsimulates at least part of the structure of APJ and that inhibits HIVentry into a target cell expressing CD4 and APJ polypeptides or thatinhibits cell-cell fusion between a first cell type that expresses CD4and APJ polypeptides and a second cell type that expresses the envprotein. The env protein of certain HIV isolates may participate in HIVinfectivity by binding to APJ at the cell surface. While not wishing tobe bound by a particular theory of the invention, the inventors believethat APJ variants may interfere in HIV infectivity by competing with APJin binding to env.

[0056] In one embodiment, the present invention relates to APJ variantsthat are peptides and peptide derivatives that have fewer amino acidresidues than APJ. Such peptides and peptide derivatives could representresearch and diagnostic tools in the study of HIV infection and thedevelopment of more effective anti-HIV therapeutics. Peptides andpeptide derivatives of APJ, according to the present invention, includethose which correspond to the extracellular regions of APJ, e.g. thefirst extracellular region (amino acids 1-25), the second extracellularregion (amino acids 91-103), the third extracellular region (amino acids168-198), or the fourth extracellular region (amino acids 272-284), asshown in FIG. 9. Peptides that correspond to the extracellular loops ofanother HIV coreceptor, the CCR5 coreceptor, have previously been shownto inhibit fusion between cells expressing the HIV-1 env and murinecells co-expressing CD4 and CCR5 (Combadiere et al., PCT/US97/09586,publication number WO 97/45543). Preferred peptides and peptidederivatives are those which correspond to a portion of either the firstextracellular region or the second extracellular region. Particularlypreferred peptides or peptide derivatives are those which comprise theAsn-Tyr-Tyr-Gly (SEQ ID NO: 3) amino acid sequence contained within thefirst extracellular region.

[0057] APJ variants useful for the present invention comprise analogs,homologs, muteins and mimetics of APJ. The variants can be generateddirectly from APJ itself by chemical modification, by proteolytic enzymedigestion, or by combinations thereof. Additionally, genetic engineeringtechniques, as well as methods of synthesizing polypeptides directlyfrom amino acid sequences, can also be employed.

[0058] Peptides of the invention can be synthesized by such commonlyused methods as t-BOC or FMOC protection of alpha-amino groups. Bothmethods involve stepwise syntheses whereby a single amino acid is addedat each step starting from the C terminus of the peptide (See, Coligan,et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit9). Peptides of the invention can also be synthesized by the well knownsolid phase peptide synthesis methods (described in Merrifield, J. Am.Chem. Soc., 85:2149 (1962) and Stewart and Young, Solid Phase PeptideSynthesis (Freeman, San Francisco, 1969) pp. 27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. The crude material cannormally be purified by standard techniques such as, for example, by gelfiltration on Sephadex G-15 using 5% acetic acid as a solvent.Lyophilization of appropriate fractions of the column will yield thehomogeneous peptide or peptide derivatives, which can then becharacterized by such standard techniques as amino acid analysis, thinlayer chromatography, high performance liquid chromatography,ultraviolet absorption spectroscopy, molar rotation, solubility, andquantitated by solid phase Edman degradation analysis.

[0059] Alternatively, peptides can be produced by recombinant methodswhich are well known to those of skill in the art.

[0060] The term “substantially purified”, as used herein, refers to amolecule, such as a peptide that is substantially free of otherproteins, lipids, carbohydrates, nucleic acids, and other biologicalmaterials with which it is naturally associated. For example, asubstantially pure molecule, such as a polypeptide, can be at least 60%,by dry weight, the molecule of interest. One skilled in the art canpurify APJ peptides using standard protein purification methods and thepurity of the polypeptides can be determined using standard methodsincluding, e.g., polyacrylamide gel electrophoresis (e.g., SDS-PAGE),column chromatography (e.g., high performance liquid chromatography(HPLC), and amino-terminal amino acid sequence analysis.

[0061] Non-peptide compounds that mimic the binding and function of APJ(“mimetics”) can be produced by the approach outlined in Saragovi etal., Science 253:792-95 (1991). Mimetics are molecules which mimicelements of protein secondary structure. See, for example, Johnson etal., Peptide Turn Mimetics, in Biotechnology and Pharmacy, Pezzuto etal., Eds., (Chapman and Hall, New York 1993). The underlying rationalebehind the use of peptide mimetics is that the peptide backbone ofproteins exists chiefly to orient amino acid side chains in such a wayas to facilitate molecular interactions. For the purposes of the presentinvention, appropriate mimetics can be considered to be the equivalentof APJ itself.

[0062] Longer peptides can be produced by the “native chemical” ligationtechnique which links together peptides (Dawson, et al., Science 266:776(1994)). Variants can be created by recombinant techniques employinggenomic or cDNA cloning methods. Site-specific and region-directedmutagenesis techniques can be employed. See Current Protocols inMolecular Biology, Vol. 1, ch. 8 (Ausubel et al. eds., J. Wiley & Sons1989 & Supp. 1990-93); Protein Engineering (Oxender & Fox eds., A. Liss,Inc. 1987). In addition, linker-scanning and PCR-mediated techniques canbe employed for mutagenesis. See PCR Technology (Erlich ed., StocktonPress 1989); Current Protocols in Molecular Biology, Vols. 1 & 2, supra.Protein sequencing, structure and modeling approaches for use with anyof the above techniques are disclosed in Protein Engineering, loc. cit.,and Current Protocols in Molecular Biology, Vol. 1 & 2, supra.

[0063] If the compounds described above are employed, the skilledartisan can routinely ensure that such compounds are amenable for usewith the present invention in view of the cell-cell fusion assay systemsand the infectivity assay systems described herein.

[0064] The invention also includes various pharmaceutical compositionsthat inhibit HIV entry into a target cell expressing CD4 and APJpolypeptides. The pharmaceutical compositions according to the inventionare prepared by bringing an APJ variant or an antibody against APJ,according to the present invention, into a form suitable foradministration to a subject using carriers, excipients and additives orauxiliaries. Frequently used carriers or auxiliaries include magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars, talc,milk, protein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial agents, anti-oxidants, chelating agents and inert gases.Other pharmaceutically acceptable carriers include aqueous solutions,non toxic excipients, including salts, preservatives, buffers and thelike, as described for instance, in Remington's Pharmaceutical Sciences,15th ed. Easton: Mack Publishing Co., 1405-1412, 1461-1487 (1975), thecontents of which are hereby incorporated by reference. The pH and exactconcentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman 's The Pharmacological Basis for Therapeutics (7thed.).

[0065] In another embodiment, the invention relates to a method ofinhibiting HIV entry into a target cell. This method involvesadministering to a subject a therapeutically effective dose of apharmaceutical composition containing the compounds of the presentinvention and a pharmaceutically acceptable carrier. “Administering” thepharmaceutical composition of the present invention may be accomplishedby any means known to the skilled artisan. By “subject” is meant anymammal, preferably a human. For example, neuropathy has been observed inthe brains of newborn infants that are born to HIV-1 seropositivemothers (Kolson et al., 1998, Adv. Virus Res. 50:1-47). Therefore, aparticularly preferred method is a method of treating a fetal subjecthaving or at risk of having an HIV infection by the administration of ananti-APJ antibody or an APJ peptide fragment. The anti-APJ antibody andthe APJ peptide fragment are preferably administered to the fetalsubject via administration to the mother.

[0066] The pharmaceutical compositions are preferably prepared andadministered in dose units. Solid dose units are tablets, capsules andsuppositories. For treatment of a subject, depending on activity of thecompound, manner of administration, nature and severity of the disorder,age and body weight of the patient, different daily doses are necessary.Under certain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

[0067] The pharmaceutical compositions according to the invention are ingeneral administered topically, intravenously, orally, or parenterallyor as implants, but even rectal use is possible in principle. Suitablesolid or liquid pharmaceutical preparation forms are, for example,granules, powders, tablets, coated tablets, (micro)capsules,suppositories, syrups, emulsions, suspensions, creams, aerosols, dropsor injectable solution in ampule form and also preparations withprotracted release of active compounds, in whose preparation excipientsand additives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, flavorings, sweeteners orsolubilizers are customarily used as described above. The pharmaceuticalcompositions are suitable for use in a variety of drug delivery systems.For a brief review of present methods for drug delivery, see Langer,Science 249:1527-1533 (1990), which is incorporated herein by reference.

[0068] The pharmaceutical compositions according to the invention may beadministered locally or systemically. By “therapeutically effectivedose” is meant the quantity of a compound according to the inventionnecessary to prevent, to cure or at least partially arrest the symptomsof the disease and its complications. Amounts effective for this usewill, of course, depend on the severity of the disease and the weightand general state of the subject. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof the pharmaceutical composition, and animal models may be used todetermine effective dosages for treatment of particular disorders.Various considerations are described, e.g., in Gilman et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th ed., Pergamon Press; and Remington 's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa., each of which is hereinincorporated by reference.

[0069] Another preferred embodiment of this invention is in thediagnosis of susceptibility to HIV infection. Nucleotide sequencesencoding the APJ receptor and antibodies to the APJ receptor can beparticularly useful for diagnosis of susceptibility to infection wherehigher levels of the receptors indicate an increased risk for HIVinfection. For example, higher levels of the APJ receptor in tissues ofthe central nervous system may indicate an increased risk ofneuropathogenesis associated with HIV infection.

[0070] Using any suitable technique known in the art, such as Northernblotting, quantitative PCR, etc., the nucleotide sequences of thereceptor or fragments thereof can be used to measure levels of APJ RNAexpression.

[0071] Alternatively, antibodies to APJ can be used in standardtechniques such as Western blotting to detect the presence of cellsexpressing the APJ receptor and in standard techniques, e.g. FACS orELISA, to quantify the level of expression. For any biological tissuesample, a level of APJ expression that is greater than a reference levelis indicative of increased susceptibility to HIV infection. A referencelevel may be established by surveying a large population of individuals.

[0072] In a preferred embodiments, the invention relates to methods forscreening a compound (“test compound”) for anti-HIV pharmacologicalactivity.

[0073] In one embodiment, a cell fusion assay is used to screen for acompound with anti-HIV pharmacological activity. In the cell fusionassay, one type of eukaryotic cell that coexpresses APJ and CD4polypeptides is incubated with a second type of eukaryotic cell thatexpresses an HIV envelope protein (“env”). Fusion between the twodifferent cell types is then monitored. The test compound is added tothe incubation solution before or after mixing of the cells and itseffect on the fusion rate of cells is determined by any number of means,including through morphological observation or through the use of anindicator system in conjunction with the cell fusion assay. Indicatorsystems useful in conjunction with a cell fusion assay can be anycombination of elements wherein a detectable signal is produced when afirst component in a first cell is brought into contact with a secondcomponent in a second cell by cell-cell fusion. For example, the firstcomponent may be a gene encoding a polymerase such as T7 polymerase andthe second component may be a gene encoding a reporter molecule which isunder the control of the T7 promoter, such as a luciferase gene. Forexample, a system that results in the production of an activeβ-galactosidase reporter molecule upon cell fusion is also contemplated.

[0074] In another embodiment, an infectivity assay is used to screen fora compound with anti-HIV pharmacological activity. In an infectivityassay, a target eukaryotic cell that expresses APJ and CD4 polypeptidesis incubated with a test virus expressing an HIV env protein. Infectionof the target cell with the test virus is then monitored. The testcompound is added to the incubation solution before or after mixing ofthe target cells with the test virus, and the effect of the compound onthe infection rate of target cells is determined by any number of means.The test virus may be a reporter virus in which the env protein ispseudotyped onto a reporter virus background. Alternatively, the testvirus may be an intact HIV virus. Infectivity is generally monitored byuse of an indicator system in conjunction with the infectivity assay.Any number of indicator systems may be used, and indicator systems whichproduce a detectable signal upon infection are preferred. For example,the reporter virus may be constructed with a gene encoding a reportermolecule such as luciferase and β-galactosidase, which is expressed whenthe reporter virus infects the target cell. As another example, thetarget cell may contain a gene encoding a reporter molecule under thecontrol of the LTR promoter, thereby resulting in expression of thereporter molecule upon infection of the target cell with the HIV virus.Alternatively, viral infection may be monitored through the use of a PCRto detect viral sequences within the infected cell.

[0075] The cell fusion assay and the HIV infectivity assay can be usedto determine the functional ability of APJ to confer env-mediated fusioncompetence to a diverse range of CD4 positive cell types (eitherrecombinantly produced or naturally occurring), including but notlimited to NIH 3T3 (murine), BS-C-1 (African green monkey), HEK 293(human), Mv1Lu (mink), U-87 MG glioblastoma, SCL1, and QT6 (quail). HIVstrains that may be used in conjunction with the assays, or as sourcesfor env protein genes to be used in conjunction with the assays, includeM-tropic, T-tropic and dual tropic strains. For example, the 89.6 dualtropic strain, the JRFL M-tropic strain, and the IIIB T-tropic HIV-1strains may be employed (Matthews et al., 1986, Proc. Natl. Acad. Sci.USA 83:9709-9713; Collman et al., 1992, J. Virol. 66:7517-7521; Gartneret al., 1986, Science 233:215-219). Additionally, selected primaryisolates may also be employed.

[0076] Variations of drug screening methods are known to the artisan ofaverage skill in this field. Consequently, the cell fusion assay and theHIV infectivity assay can be used in a wide variety of formats toexploit the properties of the APJ receptor to screen for drugs that areeffective against HIV.

[0077] Another embodiment of the invention employs the use of antisensetechnology as a specific and potent means of inhibiting HIV infection ofcells that contain APJ, for example, by decreasing the amount of APJexpression in a cell. Antisense polynucleotides in context of thepresent invention includes both short sequences of DNA known asoligonucleotides of usually 10-50 bases in length as well as longersequences of DNA that may exceed the length of the APJ gene sequenceitself. Antisense polynucleotides useful for the present invention arecomplementary to specific regions of a corresponding target mRNA.Hybridization of antisense polynucleotides to their target transcriptscan be highly specific as a result of complementary base pairing. Thecapability of antisense polynucleotides to hybridize is affected by suchparameters as length, chemical modification and secondary structure ofthe transcript which can influence polynucleotide access to the targetsite. See Stein et al., Cancer Research 48:2659 (1988). An antisensepolynucleotide can be introduced to a cell by introducing a DNA segmentthat codes for the polynucleotide into the cell such that thepolynucleotide is made inside the cell. An antisense polynucleotide canalso be introduced to a cell by adding the polynucleotide to theenvironment of the cell such that the cell can take up thepolynucleotide directly. The latter route is preferred for the shorterpolynucleotides of up to about 20 bases in length.

[0078] In selecting the preferred length for a given polynucleotide, abalance must be struck to gain the most favorable characteristics.Shorter polynucleotides such as 10-mers to 15-mers, while offeringhigher cell penetration, have lower gene specificity. In contrast, whilelonger polynucleotides of 20-30 bases offer better specificity, theyshow decreased uptake kinetics into cells. See Stein et al.,Phosphorothioate Oligodeoxynucleotide Analogues inOligodeoxynucleotides-Antisense Inhibitors of Gene Expression (Cohen,ed., McMillan Press, London 1988). Accessibility to mRNA targetsequences also is of importance and, therefore, loop-forming regions intargeted mRNAs offer promising targets.

[0079] In this disclosure, the term “polynucleotide” encompasses botholigomeric nucleic acid moieties of the type found in nature, such asthe deoxyribonucleotide and ribonucleotide structures of DNA and RNA,and man-made analogues which are capable of binding to nucleic acidsfound in nature. The polynucleotides of the present invention can bebased upon ribonucleotide or deoxyribonucleotide monomers linked byphosphodiester bonds, or by analogues linked by methyl phosphonate,phosphorothioate, or other bonds. They may also comprise monomermoieties which have altered base structures or other modifications, butwhich still retain the ability to bind naturally occurring DNA and RNAstructures. Such polynucleotides may be prespared by methods wellwell-known in the art, for instance using commercially availablemachines and reagents available from Perkin-Elmer/Applied Biosystems(Foster City, Calif.).

[0080] Phosphodiester-linked polynucleotides are particularlysusceptible to the action of nucleases in serum or inside cells, andtherefore in a preferred embodiment the polynucleotides of the presentinvention are phosphorothioate or methyl phosphonate-linked analogues,which have been shown to be nuclease resistant. Persons of ordinaryskill in this art will be able to select other linkages for use in theinvention. These modifications also may be designed to improve thecellular uptake and stability of the polynucleotides.

[0081] In another embodiment of the invention, the antisensepolynucleotide is an RNA molecule produced by introducing an expressionconstruct into the target cell. The RNA molecule thus produced is chosento have the capability to hybridize to APJ mRNA. Such molecules thathave this capability can inhibit translation of the APJ mRNA and therebyinhibit the ability of HIV to infect cells that contain the RNAmolecule.

[0082] The polynucleotides which have the capability to hybridize withmRNA targets can inhibit expression of corresponding gene products bymultiple mechanisms. In “translation arrest”, the interaction ofpolynucleotides with target mRNA blocks the action of the ribosomalcomplex and, hence, prevents translation of the messenger RNA intoprotein. Haeuptle et al., Nucl. Acids. Res. 14:1427 (1986). In the caseof phosphodiester or phosphorothioate DNA polynucleotides, intracellularRNase H can digest the targeted RNA sequence once it has hybridized tothe DNA oligomer. Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011(1988). As a further mechanism of action, in “transcription arrest” itappears that some polynucleotides can form “triplex,” or triple-helicalstructures with double stranded genomic DNA containing the gene ofinterest, thus interfering with transcription by RNA polymerase.Giovannangeli et al., Proc. Natl. Acad. Sci. 90:10013 (1993); Ebbinghauset al., J. Clin. Invest. 92:2433 (1993).

[0083] In one preferred embodiment, APJ polynucleotides are synthesizedaccording to standard methodology. Phosphorothioate modified DNApolynucleotides typically are synthesized on automated DNA synthesizersavailable from a variety of manufacturers. These instruments are capableof synthesizing nanomole amounts of polynucleotides as long as 100nucleotides. Shorter polynucleotides synthesized by modem instrumentsmay be purified by polyacrylamide gel electrophoresis or reverse phasechromatography. See Sambrook et al., Molecular Cloning: A LaboratoryManual, Vol. 2, Chapter 11, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

[0084] Alternatively, a APJ polynucleotide in the form of antisense RNAmay be introduced to a cell by its expression within the cell from astandard DNA expression vector. APJ DNA antisense sequences can becloned from standard plasmids into expression vectors, which expressionvectors have characteristics permitting higher levels of, or moreefficient expression of the resident polynucleotides. At a minimum,these constructs require a prokaryotic or eukaryotic promoter sequencewhich initiates transcription of the inserted DNA sequences. A preferredexpression vector is one where the expression is inducible to highlevels. This is accomplished by the addition of a regulatory regionwhich provides increased transcription of downstream sequences in theappropriate host cell. See Sambrook et al., Molecular Cloning: ALaboratory Manual Vol. 3, Chapter 16, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

[0085] For example, APJ antisense expression vectors can be constructedusing the polymerase chain reaction (PCR) to amplify appropriatefragments from single-stranded cDNA of a plasmid such as pRc in whichAPJ cDNA has been incorporated. Fang et al., J. Biol. Chem.267:25889-25897 (1992). Polynucleotide synthesis and purificationtechniques are described in Sambrook et al. and Ausubel et al. (eds.),Current Protocols in Molecular Biology (Wiley Interscience 1987)(hereafter “Ausubel”), respectively. The PCR procedure is performed viawell-known methodology. See, for example, Ausubel, and Bangham, ThePolymerase Chain Reaction: Getting Started, in Protocols in HumanMolecular Genetics (Humana Press 1991). Moreover, PCR kits can bepurchased from companies such as Stratagene Cloning Systems (La Jolla,Calif.) and Invitrogen (San Diego, Calif.).

[0086] The products of PCR are subcloned into cloning vectors. In thiscontext, a “cloning vector” is a DNA molecule, such as a plasmid, cosmidor bacteriophage, that can replicate autonomously in a host prokaryoticcell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Suitable cloning vectors aredescribed by Sambrook et al., Ausubel, and Brown (ed.), MolecularBiology Labfax (Academic Press 1991). Cloning vectors can be obtained,for example, from GIBCO/BRL (Gaitherburg, Md.), Clontech Laboratories,Inc. (Palo Alto, Calif.), Promega Corporation (Madison, Wis.),Stratagene Cloning Systems (La Jolla, Calif.), Invitrogen (San Diego,Calif.), and the American Type Culture Collection (Rockville, Md.).

[0087] Preferably, the PCR products are ligated into a “TA” cloningvector. Methods for generating PCR products with a thymidine or adenineoverhang are well-known to those of skill in the art. See, for example,Ausubel at pages 15.7.1-15.7.6. Moreover, kits for performing TA cloningcan be purchased from companies such as Invitrogen (San Diego, Calif.).

[0088] Cloned antisense fragments are amplified by transformingcompetent bacterial cells with a cloning vector and growing thebacterial host cells in the presence of the appropriate antibiotic. See,for example, Sambrook et al., and Ausubel. PCR is then used to screenbacterial host cells for APJ antisense orientation clones. The use ofPCR for bacterial host cells is described, for example, by Hoffman etal., Sequencing DNA Amplified Directly from a Bacterial Colony, in PCRProtocols: Methods And Applications, White (ed.), pages 205-210 (HumanaPress 1993), and by Cooper et al., PCR-Based Full-Length cDNA CloningUtilizing the Universal-Adaptor/Specific DOS Primer-Pair Strategy, Id.at pages 305-316.

[0089] Cloned antisense fragments are cleaved from the cloning vectorand inserted into an expression vector. For example, HindIII and XbaIcan be used to cleave the antisense fragment from TA cloning vectorpCR™-II (Invitrogen; San Diego, Calif.). Suitable expression vectorstypically contain (1) prokaryotic DNA elements coding for a bacterialorigin of replication and an antibiotic resistance marker to provide forthe amplification and selection of the expression vector in a bacterialhost; (2) DNA elements that control initiation of transcription, such asa promoter; and (3) DNA elements that control the processing oftranscripts, such as a transcription termination/polyadenylationsequence.

[0090] For a mammalian host, the transcriptional and translationalregulatory signals preferably are derived from viral sources, such asadenovirus, bovine papilloma virus, simian virus, or the like, in whichthe regulatory signals are associated with a particular gene which has ahigh level of expression. Suitable transcriptional and translationalregulatory sequences also can be obtained from mammalian genes, such asactin, collagen, myosin, and metallothionein genes.

[0091] Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Mol. Appl. Genes. 1:273 (1982)); the TK promoterof Herpes virus (McKnight, Cell 31:355 (1982)); the SV40 early promoter(Benoist et al., Nature 290:304 (1981); the Rous sarcoma virus promoter(Gorman et al., Proc. Nat'l. Acad. Sci. USA, 79:6777 (1982)); and thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)).

[0092] Alternatively, a prokaryotic promoter, such as the bacteriophageT3 RNA polymerase promoter, can be used to control fusion geneexpression if the prokaryotic promoter is regulated by a eukaryoticpromoter. Zhou et al., Mol. Cell. Biol. 10:4529 (1990); Kaufman et al.,Nucl. Acids Res. 19:44-85 (1991).

[0093] A vector for introducing at least one antisense polynucleotideinto a cell by expression from a DNA is the vector pRc/CMV (Invitrogen,San Diego, Calif.), which provides a high level of constitutivetranscription from mammalian enhancer-promoter sequences. Cloned APJantisense vectors are amplified in bacterial host cells, isolated fromthe cells, and analyzed as described above.

[0094] Another possible method by which antisense sequences may beexploited is via gene therapy. Virus-like vectors, usually derived fromretroviruses, may prove useful as vehicles for the importation andexpression of antisense constructs in human cells. Generally, suchvectors are non-replicative in vivo, precluding any unintended infectionof non-target cells. In such cases, helper cell lines are provided whichsupply the missing replicative functions in vivo, thereby permittingamplification and packaging of the antisense vector. A furtherprecaution against accidental infection of non-target calls involves theuse of target cell-specific regulatory sequences. When under the controlof such sequences, antisense constructs would not be expressed in normaltissues.

[0095] Two prior studies have explored the feasibility of usingantisense polynucleotides to inhibit the expression of a heparin bindinggrowth factor. Kouhara et al., Oncogene 9:455-462 (1994); Morrision, J.Biol. Chem. 266:728 (1991). Kouhara et al. showed thatandrogen-dependent growth of mouse mammary carcinoma cells (SC-3) ismediated through induction of androgen-induced, heparin binding growthfactor (AIGF). An antisense 15-mer corresponding to the translationinitiation site of AIGF was measured for its ability to interfere withandrogen-induction of SC-3 cells. At concentrations of 5 μM, theantisense polynucleotide-effectively inhibited DNA synthesis. Morrisionshowed that antisense polynucleotides targeted against basic fibroblastgrowth factor can inhibit growth of astrocytes in culture. Thus, thegeneral feasibility of targeting an individual gene product in amammalian cell has been established.

[0096] Antisense polynucleotides according to the present invention arederived from any portion of the open reading frame of the APJ cDNA.Preferably, mRNA sequences (i) surrounding the translation initiationsite and (ii) forming loop structures are targeted. Based upon the sizeof the human genome, statistical studies show that a DNA segmentapproximately 14-15 base pairs long will have a unique sequence in thegenome. To ensure specificity of targeting APJ RNA, therefore, it ispreferred that the antisense polynucleotides are at least 15 nucleotidesin length. Thus, the shortest polynucleotides contemplated by thepresent invention encompass nucleotides corresponding to positions 1-14,1-15, 1-16, 1-17, 1-18, 1-19, 2-16, 3-17, etc. of the APJ cDNA sequence.Position 1 refers to the first nucleotide of the APJ coding region.

[0097] Not every antisense polynucleotide will provide a sufficientdegree of inhibition or a sufficient level of specificity for the APJtarget. Thus, it will be necessary to screen polynucleotides todetermine which have the proper antisense characteristics. A preferredmethod to assay for a useful antisense polynucleotide is the inhibitionof cell fusion between (1) cells that contain CD4 and APJ; and (2) cellsthat contain env.

[0098] Administration of an antisense polynucleotide to a subject,either as a naked, synthetic polynucleotide or as part of an expressionvector, can be effected via any common route (oral, nasal, buccal,rectal, vaginal, or topical), or by subcutaneous, intramuscular,interperitoneal, or intravenous injection. Pharmaceutical compositionsof the present invention, however, are advantageously administered inthe form of injectable compositions. A typical composition for suchpurpose comprises a pharmaceutically acceptable solvent or diluent andother suitable, physiologic compounds. For instance, the composition maycontain polynucleotide and about 10 mg of human serum albumin permilliliter of a phosphate buffer containing NaCl.

[0099] As much as 700 milligrams of antisense polynucleotide has beenadministered intravenously to a patient over a course of 10 days (i.e.,0.05 mg/kg/hour) without signs of toxicity. Sterling, “SystemicAntisense Treatment Reported,” Genetic Engineering News 12:1, 28 (1992).

[0100] Other pharmaceutically acceptable excipients include non-aqueousor aqueous solutions and non-toxic compositions including salts,preservatives, buffers and the like. Examples of non-aqueous solutionsare propylene glycol, polyethylene glycol, vegetable oil and injectableorganic esters such as ethyloleate. Aqueous solutions include water,alcoholic/aqueous solutions, saline solutions, parenteral vehicles suchas sodium chloride, Ringer's dextrose, etc. Intravenous vehicles includefluid and nutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to routine skills in the art. A preferredpharmaceutical composition for topical administration is a dermal creamor transdermal patch.

[0101] Antisense polynucleotides or their expression vectors may beadministered by injection as an oily suspension. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate or triglycerides.Moreover, antisense polynucleotides or vectors may be combined with alipophilic carrier such as any one of a number of sterols includingcholesterol, cholate and deoxycholic acid. A preferred sterol ischolesterol. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension include, for example, sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. Optionally, thesuspension also contains stabilizers.

[0102] An alternative formulation for the administration of antisenseAPJ polynucleotides involves liposomes. Lipsome encapsulation providesan alternative formulation for the administration of antisense APJpolynucleotides and expression vectors. Liposomes are microscopicvesicles that consist of one or more lipid bilayers surrounding aqueouscompartments. See, generally, Bakker-Woudenberg et al., Eur. J. Clin.Microbiol. Infect. Dis. 12 (Suppl. 1): S61 (1993), and Kim, Drugs 46:618(1993). Lipsomes are similar in composition to cellular membranes and asa result, liposomes can be administered safety and are biodegradable.Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and liposomes can vary in size with diameters rangingfrom 0.01 μm to greater than 10 μm. A variety of agents can beencapsulated in liposomes: hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s). See,for example, Machy et al., Liposomes in Cell Biology And Pharmacology(John Libby 1987), and Ostro et al., American J. Hosp. Pharm. 46:1576(1989). Moreover, it is possible to control the therapeutic availabilityof the encapsulated agent by varying liposome size, the number ofbilayers, lipid composition, as well as the charge and surfacecharacteristics of the liposomes.

[0103] Liposomes can adsorb to virtually any type of cell then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents. Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985).

[0104] After intravenous administration, conventional liposomes arepreferentially phagocytosed into the reticuloendothelial system.However, the reticuloendothelial system can be circumvented by severalmethods including saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means. Claassen etal., Biochim. Biophys. Acta 802:428 (1984). In addition, incorporationof glycolipid- or polyethelene glycol-derivatised phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system. Allen et al., Biochim.Biophys. Acta. 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993). These Stealth® liposomes have an increased circulationtime and an improved targeting to tumors in animals. Woodle et al.,Proc. Ameri. Assoc. Cancer Res. 33:2672 (1992); Gregoriadis et al.,Drugs 45:15 (1993).

[0105] Antisense polynucleotides and expression vectors can beencapsulated within liposomes using standard techniques. A variety ofdifferent liposome compositions and methods for synthesis are known tothose of skill in the art. See, for example, U.S. Pat. No. 4,844,904,U.S. Pat. No. 5,000,959, U.S. Pat. No. 4,863,740, and U.S. Pat. No.4,975,282, all of which are hereby incorporated by reference.

[0106] Liposomes can be prepared for targeting to particular cells ororgans by varying phospholipid composition or by inserting receptors orligands into the liposomes. For instance, antibodies specific to tumorsassociated with antigens may be incorporated into liposomes, togetherwith antisense polynucleotides or expression-vectors, to target theliposome more effectively to the tumor cells. See, for example, Zelphatiet al., Antisense Research and Development 3:323-338 (1993), describingthe use “immunoliposomes” containing antisense polynucleotides for humantherapy.

[0107] In general, the dosage of administered liposome-encapsulatedantisense polynucleotides and vectors will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. Dose ranges for particularformulations can be determined by using a suitable animal model.

[0108] The above approaches can also be used not only with antisensenucleic acids, but also with ribozymes, or triplex agents to blocktranscription or translation of a specific APJ mRNA, either by maskingthat mRNA with an antisense nucleic acid or triplex agent, or bycleaving it with a ribozyme.

[0109] Use of an oligonucleotide to stall transcription is known as thetriplex strategy since the oligomer winds around double-helical DNA,forming a three-strand helix. Therefore, these triplex compounds can bedesigned to recognize a unique site on a chosen gene (Maher, et al.,Antisense Res. and Dev., 1(3):227, 1991; Helene, C., Anticancer DrugDesign, 6(6):569, 1991).

[0110] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

[0111] There are two basic types of ribozymes namely, tetrahymena-type(Hassellhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-base recognition sequences are preferable to shorter recognitionsequences.

[0112] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The following examples are to beconsidered illustrative and thus are limiting to the remainder of thedisclosure.

EXAMPLES Example 1 A Cell-Cell Fusion Assay to Determine Whether APJCould Function as a Coreceptor for HIV-1 or SIV

[0113] A cell-cell fusion assay was employed to determine whether APJcould function as a coreceptor for HIV-1 or SIV. This assay has beendescribed in detail in Nussbaum et al., 1994, J. Virol., 68:5411-5422and in Rucker et al., 1997, Meth. Enzymol., 288:118-133. Effector cellswere prepared by infecting quail QT6 cells with a recombinant vacciniavirus encoding T7 polymerase (vTF 1.1) and then either transfecting thecells with a plasmid bearing the envelope gene of interest under thecontrol of the T7 promoter or introducing the env constructs viarecombinant vaccinia virus. Env constructs SIVmac251, SIVmac239,SIVmac316, SIVmac316mut, DH12, RF, BK132, ADA, JR-FL, ITIB and HIV-2 STwere introduced into effector cells via recombinant vaccinia virusrather than by transfection. QT6 target cells were prepared by transienttransfection with plasmids encoding CD4, the coreceptor of interestunder the control of the CMV promoter, and luciferase under the controlof the T7 promoter. Effector and target cells were mixed the day aftertransfection and cell-cell fusion was quantified by measuring luciferaseactivity in cell lysates 7-8 hours following mixing.

[0114] In this assay, cell-cell fusion results in cytoplasmic mixing andluciferase production, which can be easily quantified. As shown in FIG.1, co-expression of either the CCR5 or the CXCR4 coreceptor with CD4resulted in efficient fusion by R5 and X4 Env proteins, respectively.R5-X4 env proteins such as HIV-1 89.6 mediated fusion with cells bearingeither the CCR5 or the CXCR4 coreceptor. Fusion was not observed whenCD4 was expressed alone. When APJ was co-expressed with CD4 in QT6cells, cell-cell fusion was mediated by the R5-X4 Env protein 89.6 andby several X4 env proteins at levels≧70% of that observed with CXCR4(FIG. 1). For one primary X4 env protein, ZR001.3, fusion with cellsexpressing APJ was more efficient than with CXCR4. A majority of R5 envproteins mediated fusion with APJ expressing cells, but only at very lowlevels relative to that observed with CCR5 (FIG. 1 and Table 1).However, ADA and the primary isolate TH 22-4 exhibited fusion mediatedby APJ at roughly half the level observed when CCR5 served as the viralcoreceptor. The HIV-2 ST env protein also mediated very inefficientfusion with cells expressing both CD4 and APJ. The ability of APJ tosupport fusion for some X4 and R5-X4 viral env proteins nearly asefficiently as the major coreceptors is notable, since most otheralternative HIV-1 coreceptors typically support cell-cell fusion muchless efficiently than CCR5 or CXCR4.

[0115] The ability of APJ to support fusion by a panel of SIV envelopeproteins was also examined. Unlike HIV-1, both M- and T-tropic SIVstrains utilize CCR5 as a coreceptor, while CXCR4 is either not used orrarely used by SIV (Chen et al., 1997, J. Virol., 71:2705-2714; Edingeret al., 1997, Proceedings of the National Academy of Sciences, USA,94:4005-4010; and Marcon et al., 1997, J. Virol., 71:2522-2527). Inaddition, the orphan receptors STRL33, GPR15, and GPR1 can be used ascoreceptors by both T- and M-tropic SIV strains (Deng et al., 1997,Nature, 388:296-300 and Farzan et al., 1997, J. Exp. Med., 186:405-411).In the instant experiments, it was determined that APJ supported fusionby several M- and T-tropic SIV env proteins at levels that were lessefficient than those observed with CCR5, with the exception of theM-tropic SIVmac316 and a variant of this env protein (316mut) whichefficiently used APJ as a coreceptor in cell-cell fusion assays (FIG. 2and Table 1). Additionally, APJ typically supported fusion lessefficiently than the orphan receptors GPR1, GPR15/BOB, and STRL33/Bonzo.Finally, because it was previously determined that many SIV strains caninfect cells in a CD4-independent, CCR5-dependent manner (Edinger etal., 1997, Proc. Natl. Acad. Sci., USA, 94:14742-14747), the ability ofHIV-1, HIV-2, and SIV env proteins to mediate fusion with cellsexpressing APJ alone was tested. The results showed that APJ coreceptoractivity was strictly CD4 dependent, as cells expressing APJ alone didnot support cell-cell fusion with any of the env proteins tested. TABLE1 ENV Tropism CCR5 CXCR4 APJ DH12 D +++ +++ + RF (D) +++ +++ − YU2 M +++− + JR-FL M +++ − − SF162 M +++ − + 91US005.11 +++ − + 93BR019.10 +++− + 92UG031.7 +++ − − 93BR029.2 +++ − − UG37-8 +++ − + TH 22-4 +++ − ++RW20-5 +++ − + SIVmacBK28 +++ − + SIV/17E-C1 M +++ − + SIVmac1A11 M +++− + SIVagmSab1.4 +++ − + SIVsm62A T +++ − + SIVsm62D T +++ − +SIVsm543-3 M +++ − ++ SIVsm543-B10 +++ − + SIVsmPBj6 +++ − +

Example 2 Determination of APJ Ability to Support Virus Infection

[0116] The ability of APJ to support virus infection was determined inorder to more rigorously assess the ability of APJ to function as acoreceptor. A first assay system employed a luciferase reporter virusassay in which various env proteins were pseudotyped onto the luciferasereporter virus backbone. Luciferase reporter viruses were prepared bytransfecting human HEK 293 cells with a plasmid that expresses env underthe control of the CMV or SV40 promoter, and with a plasmid containing aproviral genome with an inactive env gene and the luciferase gene inplace of nef (e.g. the NL4-3 luciferase virus backbone (pNL-Luc-E-R-))(Chen et al., 1994, J. Virol., 68:654-660 and Connor et al., 1995,Virology, 206:935-944). Target cells for infection were HEK 293 or CCCScells with CD4 and coreceptors introduced by calcium phosphatetransfection. Infections were performed in media containing 8 μg/ml DEAEdextran. Cells were lysed 3-4 days post-infection by resuspension in0.5% NP-40 in PBS and assayed for luciferase activity.

[0117] Unfortunately, most env proteins which efficiently catalyzedfusion with cells expressing CD4 and APJ (such as HIV-1 89.6) could notbe successfully pseudotyped. Viral env proteins that could bepseudotyped, as judged by infection of CCR5 or CXCR4 positive cells,either failed to infect cells expressing CD4 and the APJ coreceptor ordid so inefficiently (FIG. 3). In some cases, env proteins that mediatedfusion with APJ expressing cells at intermediate levels failed tosupport virus infection. For example, the virus pseudotype with the ADAenv protein did not infect APJ-positive cells even though cellsexpressing the ADA env protein mediated fusion with APJ-positive cellshalf as efficiently as with CCR5-positive cells. The reasons for theseassay dependent discrepancies are not clear, but may reflect theefficiencies with which various env proteins can be pseudotyped.

[0118] Another assay system was employed in order to test envelopeproteins which could not be pseudotyped but which were able to mediatecell-cell fusion with APJ expressing cells in an efficient manner.Target HEK 293 cells that had been transfected with plasmids expressingCD4, the desired coreceptor, and luciferase under control of the viralLTR were infected with intact HIV-1 89.6 or HIV-1 IIIB (FIG. 4).Luciferase activity was measured 2 days-post infection. The resultsshowed that HIV-1 89.6 infected APJ positive cells nearly as efficientlyas cells expressing CXCR4. HIV-1 IIIB (HxB3) also infected APJ positivecells a levels well above background.

[0119] Finally, a PCR based entry assay was also employed to determineif APJ could support infection by HIV-1 89.6 and IIIB. QT6 cells stablyexpressing human CD4 and transiently expressing the desired coreceptorwere infected with 50 ng p24 of DNAase-treated, cell-free virus. Aftertwo days, the cells were washed and lysed, and HIV-1 specific LTR DNAsequences were detected by PCR using primers LTR-plus/LTR-minus(5′-ACAAGCTAGTACCCAGTTGAGCC-3′ (SEQ ID NO: 4),5′-CACACACTACTTGAAGCACTCA-3′ (SEQ ID NO: 5)). Products were resolved byelectrophoresis on 2% agarose gels, transferred to Hybond N+ (Amersham),and detected by using the 3′-End Labeling Biotin Kit (DuPont; probe5′-ATCTACAAGGGACTTTCCCGC-3′ (SEQ ID NO:6), followed by exposure. Asshown in FIG. 5, both HIV-1 IIIB and 89.6 could enter QT6 cellsexpressing both CD4 and APJ, although entry was less efficient than withthe major HIV-1 coreceptors.

Example 3 Examination of the of the Distribution of APJ in Human Brainby Northern Blot Analysis

[0120] APJ was originally cloned from human genomic DNA, and analysis ofrat tissues using a probe based on the rat homolog revealed that APJ isexpressed widely in brain (O'Dowd et al., 1993, Gene, 136:355-360). APJhas also been shown to be expressed in some areas of the human brain(Matsumoto et al., 1996, Neurosci. Lett., 219:119-122). Because of theefficient use of APJ as a coreceptor by some virus strains, APJdistribution in the human brain has been further examined by Northernblot analysis.

[0121] Membranes containing poly A⁺ RNA from various human brain regionswere obtained from Clontech. The Prime-It II Random Primer Labeling Kit(Stratagene, La Jolla, Calif.) was used to label the cDNA probe withα-³²P-dATP (3,000 Ci/mmol) using the Klenow enzyme. The α-³²P-labeledcDNA probe was purified using Quick Spin columns (Boehringer Mannheim,Indianapolis, Ind.). The membranes were hybridized overnight with 10⁷cpms of the labeled probe in hybridization buffer containing 25 mMNa/Na₂PO₄, 50 mM Tris pH 7.4, 6× SSPE, 0.1% SDS, 1100 μg/ml singlestranded DNA and 1× Denhardt's solution. The membranes were washed twicein 1× SSPE, 0.1% SDS at 42° C. for 10 minutes and changed to a highstringency wash solution of 0.2× SSPE, 0.1% SDS at 42° C. for 10minutes. The membrane was then exposed to a Fuji Imaging plate for 4hours. Images of the plate were captured on a BAS1000Mac Bio-ImagingAnalyzer (Fuji) and processed with Mac BAS software. Images were printedon a Pictography 3000 (Fuji) digital printer.

[0122] The results showed that high levels of APJ transcripts werepresent in the corpus callosum, spinal cord, and medulla. Lower levelsof APJ transcripts were detected in other regions of the human brain(FIG. 6). In peripheral tissues, the APJ transcript was readily detectedin spleen but absent in PBLs (FIG. 7). Lower levels of transcript weredetected in other peripheral tissues.

[0123] To investigate the distribution of APJ in cells commonly used topropagate HIV-1, RT-PCR analysis was performed on a large number of celllines and some primary cell types. A U87 cell line that stably expressedAPJ was generated and used as a positive control.

[0124] Primary cells were isolated as follows. Human blood mononuclearcells (PBMC) were isolated from blood of normal volunteers usingFicoll-Hypaque, depleted of monocytes by serial adherence to plastic,stimulated with phytohemagglutinin (PHA-L, 5 μg/ml; Sigma) for 3 daysand then resuspended with interleukin 2 (20 U/ml, Boehringer MannheimBiochemicals). RNA was extracted after 3 days of PHA stimulation andalso following 1 week in IL-2. Monocytes were purified from PBMC byselective adherence to gelatin followed by plastic, and then maintainedin culture to allow differentiation into monocyte-derived macrophages(MDM) as previously described (Collman et al., 1989, J. Exp. Med.,170:1149-1163). RNA was extracted from undifferentiated monocytesimmediately after purification and from MDM after 1 week in culture.

[0125] For the isolation of total cellular RNA for RT-PCR, 5-10×10⁶cells were resuspended in 1 ml Trizol (GIBCO-BRL) and processed asrecommended by the manufacturer. Total RNA was then treated with 1 μl(10-50 units) DNAse (RNAse-free) (Boehringer Mannheim) per 10 μg RNA for30 min at 37° C. in the presence of 5 mM MgCl₂ with subsequentinactivation at 65° C. for 10 minutes in the presence of 5 mM EDTA; RNAconcentration was calculated based on the OD₂₆₀. The Titan RT-PCR system(Boehringer Mannheim) was used to evaluate RNA expression patterns.Specific, internal upstream and downstream primers were used whichresulted in an amplified product of 481 base pairs. The primers usedwere the following: forward 5′-TACACAGACTGGAAATCCTCG-3′ (SEQ ID NO: 7)and reverse 5′-TGCACCTTAGTGGTGTTCTCC-3′ (SEQ ID NO: 8). In order tocontrol for contamination of the RNA sample with genomic DNA despitetreatment with DNAse, all RNA samples were also amplified with Titanenzyme mix in which the RT but not PCR activity had been destroyed bytreatment at 95° C. for 10 minutes (this inactivation protocol was foundto eliminate the ability to amplify a RNA but not a DNA template). Ineach RT-PCR reaction, RNA isolated from U87-APJ stably transfected cellswere included as a positive RNA control and plasmid DNA was included asa second positive control.

[0126] The results of the investigation of the distribution of APJ incell lines and cell types showed that APJ was expressed in C8166 cells,but APJ-specific reaction products could not be detected in the othercell lines exarnined, including Jurkat, Hut78, CEMx174, and PM1 cells.Additionally, expression of APJ was not detected in PHA, PHA with IL-2,or anti-CD3 and IL-2 stimulated PBMC or in monocytes or monocyte derivedmacrophages (FIG. 8).

[0127] For other aspects of the nucleic acids, polypeptides, antibodies,etc., reference is made to standard textbooks of molecular biology,protein science, and immunology. See, e.g., Davis et al. (1986), BasicMethods in Molecular Biology, Elsevir Sciences Publishing, Inc., NewYork; Hames et al. (1985), Nucleic Acid Hybridization, IL Press,Molecular Cloning, Sambrook et al.; Current Protocols in MolecularBiology, Edited by F. M. Ausubel et al., John Wiley & Sons, Inc; CurrentProtocols in Human Genetics, Edited by Nicholas C. Dracopoli et al.,John Wiley & Sons, Inc.; Current Protocols in Protein Science; Edited byJohn E. Coligan et al., John Wiley & Sons, Inc.; Current Protocols inImmunology; Edited by John E. Coligan et al., John Wiley & Sons, Inc.

[0128] The entire disclosure of all patent applications, patents, andpublications cited herein are hereby incorporated by reference.

[0129] From the foregoing description, on skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A recombinant eukaryotic cell transformed with apolynucleotide encoding an APJ polypeptide or a polynucleotide encodinga CD4 polypeptide, wherein the cell coexpresses APJ and CD4polypeptides.
 2. A recombinant eukaryotic cell transformed with apolynucleotide encoding an APJ polypeptide and a polynucleotide encodinga CD4 polypeptide, wherein the cell coexpresses APJ and CD4polypeptides.
 3. A recombinant eukaryotic cell according to claim 1,wherein the cell is stably transformed.
 4. A recombinant eukaryotic cellaccording to claim 2, wherein the cell is stably transformed bothpolynucleotides.
 5. The cell as in any of claims 1-4, wherein the cellis a human cell.
 6. The cell as in any of claims 1-4, wherein the cellis a non-human cell.
 7. An antibody which specifically binds to anextracellular domain of APJ, wherein the antibody inhibits HIV infectionof a target cell that coexpresses APJ and CD4 polypeptides.
 8. Anantibody which specifically binds to an extracellular domain of APJ,wherein the antibody inhibits membrane fusion between a first cellcoexpressing APJ and CD4 polypeptides and second cell expressing an HIVenv protein.
 9. An antibody according to claim 7 or 8, wherein theantibody is a monoclonal antibody.
 10. An antibody according to claim 9,wherein the antibody recognizes an epitope comprising an amino acidsequence corresponding to a portion of the first extracellular domain ofAPJ.
 11. An antibody according to claim 10, wherein the amino acidsequence corresponding to a portion of the first extracellular domain ofAPJ comprises the amino acid sequence Asn-Tyr-Tyr-Gly (SEQ ID NO: 3).12. An antibody according to claim 9, wherein the antibody recognizes anepitope comprising an amino acid sequence corresponding to a portion ofthe second extracellular domain of APJ.
 13. A substantially purifiedpeptide fragment of APJ, wherein the peptide inhibits HIV infection of atarget cell that coexpresses APJ and CD4 polypeptides.
 14. Asubstantially purified peptide fragment of APJ, wherein the peptideinhibits cell fusion between a first cell coexpressing APJ and CD4polypeptides and a second cell expressing an HIV env protein.
 15. Asubstantially purified peptide fragment of APJ according to claim 13 or14, wherein the peptide fragment comprises an amino acid sequencecorresponding to a portion of the first extracellular domain of APJ. 16.A substantially purified peptide fragment of APJ according to claim 15,wherein the amino acid sequence corresponding to a portion of the firstextracellular domain of APJ comprises the amino acid sequenceAsn-Tyr-Tyr-Gly (SEQ ID NO:3).
 17. A substantially purified peptidefragment of APJ according to claim 13 or 14, wherein the peptidefragment comprises an amino acid, sequence corresponding to a portion ofthe second extracellular domain of APJ.
 18. A method for identifying acompound that modulates interaction between an HIV virus and an APJreceptor comprising incubating a first cell line which coexpresses CD4and APJ polypeptides with a second cell line which expresses an envprotein under conditions which promote cell fusion, in the presence andabsence of a test compound, and determining whether the presence of thetest compound inhibits cell fusion between the first cell line and thesecond cell line.
 19. A method according to claim 18, wherein cellfusion is determined by detection of a reporter molecule.
 20. A methodaccording to claim 19, wherein the reporter molecule is selected fromthe group consisting of a radioisotope, a fluorescent compound, abioluminescent compound, a chemiluminescent compound, a metal chelator,or an enzyme.
 21. A method according to claim 19 wherein the reportermolecule is B-galactosidase or luciferase.
 22. A method for identifyinga compound that modulates interaction between an HIV virus and an APJreceptor comprising incubating a cell line which expresses CD4 and APJpolypeptides with a test virus carrying an env protein, in the presenceand absence of a test compound, and determining whether the presence ofthe test compound inhibits infection of the cell line by the test virus.23. A method according to claim 22, wherein infection is determined bydetection of a reporter molecule.
 24. A method according to claim 23,wherein the reporter molecule is selected from the group consisting of aradioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator, or an enzyme.
 25. A methodaccording to claim 23, wherein the reporter molecule is B-galactosidaseor luciferase.
 26. A method of inhibiting HIV infection of a target cellexpressing an APJ and CD4 polypeptides comprising contacting the targetcell with an effective amount of a APJ binding or blocking agent. 27.The method of claim 26, wherein the agent is an anti-APJ antibody orepitope binding fragment thereof.
 28. The method of claim 27, whereinthe antibody is a monoclonal antibody or a polyclonal antibody.
 29. Themethod of claim 26, wherein said contacting is accomplished by in vivoadministration to a subject.
 30. The method of claim 26, wherein theagent is a peptide fragment of APJ.
 31. A method of treating a subjecthaving an HIV-related disorder associated with expression of APJcomprising administering an agent that suppresses APJ to the subject.32. The method of claim 31, wherein the agent is an anti-APJ antibody.33. The method of claim 31, wherein the agent is an antisensepolynucleotide that hybridizes to an APJ polynucleotide.
 34. The methodof claim 31, wherein the agent is introduced into a cell using acarrier.
 35. The method of claim 34, wherein the carrier is a vector.36. A method of treating a subject having or at risk of having an HIVinfection or related disorder, comprising administering atherapeutically effective amount of an anti-APJ antibody or a peptidefragment to the subject.
 37. A method according to claim 36, wherein thesubject is a fetus.
 38. A transgenic non-human animal having a phenotypecharacterized by expression of APJ polypeptide and CD4 polypeptideotherwise not naturally occurring in the animal, wherein the phenotypeis conferred by a transgene contained in the somatic cells and germcells of the animal, and wherein the transgene comprises apolynucleotide encoding an APJ polypeptide and a polynucleotide encodinga CD4 polypeptide.